Microarrays are used in bioassaying for the presence and/or quantitative amount of a target material in a biological sample; they constitute a growing field that is sometimes referred as surface-based assays wherein the target material, or a molecule representative of such, is captured on a solid support and then detected. Such DNA microarrays have now become widely accepted for the study of gene expression and other genotyping functions as a result of their capacity to simultaneously monitor a large number of genes. For example, a large number of different probe sequences can be bound at distinct spatial locations or microspots across a microarray surface, and each such microspot may contain a different probe. When such a microarray is hybridized with a solution containing labeled sample material, hybridization occurs between complimentary DNA strands at the various different microspots of the array whenever the target is present. After washing to remove unbound material, the character of the labels, e.g., fluorescent, is then used to determine the intensity of labeled material at each microspot; this imaging can provide a measure of the quantitative amount of the particular target that was present in the sample.
Such arrays have also been fabricated to present other moieties, such as proteins including antibodies, and haptens or aptamers, for binding to target materials. These surface-based assays can also be used for ELISAs. Overall, the use of such microassay chips has been replacing gel electrophoresis as the method of choice for bioassaying, and this trend is believed very likely to continue as the field of proteomics becomes more advanced.
For a microassay chip to be effective for such bioassay applications, it should have the ability to immobilize a satisfactory amount of analyte or target material to be sequestered from a relevant sample; it is in this manner that a signal of satisfactory magnitude is provided when the chip is subjected to subsequent reading. The chip, of course, should also be capable of being fabricated so as to be highly uniform, in order to produce reproducible results from assay to assay.
Many microarray chips have been developed in the past decade where probes have been immobilized on a modified glass substrate, a silicon substrate, or the like, at distinct spatial locations, to create an array which presents a large number of different probes. Initially microarrays were developed as a two-dimensional form wherein probes were directly bound on the surface on the substrate. More recently three-dimensional microarrays have been developed using hydrogel materials wherein the microspots may resemble minute hemispheres, the porous structures of which present a three-dimensional framework or matrix. Microarrays of this type are described in U.S. Pat. No. 6,174,683 and in published International Application WO 02/059372.
U.S. published application 2003/0124371 discloses the use of water-swellable hydrophilic hydrogels which are considered to be particularly useful for immobilizing polypeptide analytes onto an absorbent layer, which layer is engineered by varying the ratio of hydrophilic moieties and hydrophobic moieties in the hydrogel. The hydrophilic and hydrophobic monomers which make up the hydrogel are cross-linked to create a desired polymer. As an example, an aluminum substrate is coated with silicon dioxide and then treated with an alkylsilane before the monomers are applied to a plurality of addressable locations (microspots) and then cross-linked by radiation. Probes are added to each microspot on the chip, using a binding buffer, and the loaded chip is incubated for thirty minutes. Washing then readies the chip for use in an assay.
U.S. published application 2003/0138649 teaches the fabrication of microarrays particularly suited for attaching proteins which will serve as probes or capture agents using a gelatin-based substrate. A suitable substrate such as glass or silicon or photographic paper is coated with a solution of type IV gelatin; for example, gelatins were coated onto reflective photographic paper and then chill-set and dried. The plates having the overall gelatin coating are then microspotted to attach bi-functional compounds, e.g. goat anti-mouse antibody IgG, which has a group that will link to the gelatin and a second functional group that is capable of interacting with high specificity with a protein. In a related U.S. published application, No. 2003/0170474, a silicon wafer or glass plate is treated first with an alkylsilane and then dipped in a solution of gelatin. The gelatin-coated substrate is then dipped in a solution of polyethyleneimine (PEI). The surface was reported to have a relatively low nonspecific binding capacity for proteins and that it could be used as a microarray substrate by affixing protein capture agents at microspots spaced across the surface.
U.S. published application 2003/0096257 teaches the making of DNA chips by coating a glass slide with an aminoalkylsilane and then attaching vinylsulfonyl groups across the entire surface by bonding to the amino groups. Oligonucleotides with linkers were then spotted onto the reactive plate and suitably incubated to secure the linkage to produce a DNA chip useful for a hybridization analysis.
Although there may be various advantages to these methods of making of biochips or the like, each of them is not without its disadvantages. Accordingly, the search has continued for still better methods for the fabrication of such microarrays with emphasis often being concentrated on the employment of hydrogels in such microarrays.